Truncated keratinocyte growth factor (KGF) having increased biological activity

ABSTRACT

The present invention relates to a keratinocyte growth factor fragment, KGFdes1-23, or an analog thereof that is composed of a portion of an amino acid sequence of mature, full length keratinocyte growth factor, KGF163. The fragment exhibits at least a 2-fold increase in mitogenic activity as compared to a mature, recombinant keratinocyte growth factor, rKGF, but lacks a sequence comprising the first 23 amino acid residues, C-N-D-M-T-P-E-Q-M-A-T-N-V-N-C-S-S-P-E-R-H-T-R- (SEQ ID NO: 2) of the KGF163 N-terminus. The present invention also relates to a DNA molecule encoding KGFdes1-23, an expression vector and a transformed host containing the DNA molecule, and a method of producing KGFdes1-23 by culturing the transformed host. The present invention further relates to a conjugate of KGFdes1-23 and a toxin molecule, and the use thereof for treatment of hyperproliferative disease of the epidermis. Moreover, the present invention relates to a therapeutic composition containing KGFdes1-23 and a pharmaceutically acceptable carrier and the use thereof for wound healing purposes.

This application is a continuation of application Ser. No. 08/086,427filed on 29 Jun. 1993, now abandoned.

FIELD OF THE INVENTION

The present invention relates generally to keratinocyte growth factor("KGF"). More specifically, this invention relates to a KGF fragment andanalogs thereof having increased biological activity, and decreasedcytotoxicity as compared to a mature, recombinant, full length KGFexpressed in an insect cell expression system. This KGF fragment lacksthe first 23 amino acid residues of the N-terminus of mature,full-length KGF, including a potential glycosylation site. TheN-terminus was previously believed to confer upon KGF its epithelialcell specificity.

KGF belongs to the family of fibroblast growth factors ("FGFs"), theprototype of which is represented by basic FGF ("bFGF"). KGF is, hence,also known as FGF-7. Like other FGFs, KGF is a heparin-binding protein,but unlike other FGFs, it has a unique target cell specificity. Inparticular, FGFs are generally capable of stimulating the proliferationand differentiation of a variety of cell types derived from the primaryor secondary mesoderm as well as from neuroectoderm. KGF is similar toother FGFs in its ability to stimulate epithelial cell proliferation,but is dissimilar to other FGFs in its inability to stimulateendothelial cells or fibroblast proliferation, as discussed in Finch, P.W. et al., Science 245: 752-755 (1989). Mature, full-length KGF,designated herein as KGF₁₆₃, is a polypeptide with 163 amino acidresidues, and possesses a potential N-glycosylation site at amino acid14 of the consensus sequence for glycosylation that extends from aminoacid residue 14 to 16 at the N-terminus, as indicated in Finch et al.(1989), loc. cit.

BACKGROUND OF THE INVENTION

FGFs, including acidic fibroblast growth factor ("aFGF") and basicfibroblast growth factor ("bFGF"), are known to have heparin-bindingproperties and have the ability to induce the differentiation andproliferation of ventral, as well as dorsal, mesoderm in earlyblastulae, as discussed in Gospodarowicz et al., Cell. Biol. Rev.25:307-314 (1991), and Basilico et al., Adv. Cancer Res. 59:115-165(1992). The response of cells to FGF is mediated through binding thereofto cell surface receptors known as fibroblast growth factor receptors("FGFRs"), of which there are three interrelated types, as discussed inHou et al., Science 251:665-668 (1991). High affinity FGFRs are tyrosinekinases and include the flg receptor ("FGFR-1"), the bek receptor("FGFR-2"), and the K Sam receptor ("FGFR-3"), as discussed in Lee etal., Science 245:57-60 (1989); Dionne et al., EMBO J. 9:2685-2692(1990); Miki et al., Science 251:72-75 (1991); Miki et al., Proc. Natl.Acad. Sci. USA 89:246-250 (1992); and Dell et al., J. Biol. Chem.267:21225-21229 (1992).

Both FGFR-1 and FGFR-2 are widely expressed in mesodermal andneuroectodermal tissues, and both are able to bind aFGF and bFGF withsimilar affinities. FGFR-3, also referred to as KGFR, is a KGF receptorthat is specific to epithelial cells. It is an alternative transcript ofFGFR-2. In contrast to FGFR-2, which shows high affinity for both aFGFand bFGF and no affinity for KGF, FGFR-3 binds KGF and aFGF with anaffinity approximately 20 to 1000 fold higher than bFGF, as discussed inMiki et al. (1992), and Dell et al. (1992), loc. cit.

The tightly restricted tissue distribution of KGFR to epithelial cellsand, therefore, the tissue restricted activity of KGF, is desirable inmany types of wound healing applications, as well as in the treatment ofhyperproliferative diseases of the epidermis, such as psoriasis andbasal cell carcinoma. Presently, except for KGF, no highly suitablefactor exists for these applications. It would be desirable, therefore,if KGF could be modified to increase its potency and decrease itscytotoxicity for therapeutic applications.

Recently, Ron et al., J. Biol. Chem. 268:2984-2988 (February 1993) foundthat when KGF₁₆₃ was expressed in a prokaryotic expression system, arecombinant KGF ("rKGF") polypeptide could be obtained that possessedmitogenic activity. When the rKGF molecule was truncated by deletion of3, 8, 27, 38, and 48 amino acid residues from the N-terminus of themature KGF₁₆₃ polypeptide, biological activity of the resultingmolecules varied. With deletion of 3 and 8 amino acid residues,respectively, the mitogenic activity of the resulting molecules did notappear to be affected as compared to full-length rKGF. Deletion of 27amino acid residues, however, resulted in molecules that display 10-20fold reduced mitogenic activity. Deletion of 38 and 48 amino acidresidues, respectively, resulted in complete loss of mitogenic activityand heparin-binding ability. Ron et al., however, failed to produce anytruncated rKGF fragments that possessed increased mitogenic activity ascompared to the full-length rKGF molecule.

SUMMARY OF THE INVENTION

One of the objects of the present invention is to provide a KGF fragmentor an analog thereof that contains a portion of the amino acid sequenceof mature, full-length KGF and that exhibits at least a 2-fold, butpreferably 7-fold, more preferably, a 7-10 fold increase in mitogenicactivity as compared to the mature, full-length rKGF. This KGF fragmentlacks a sequence comprising the first 23 N-terminal amino acid residues,C-N-D-M-T-P-E-Q-M-A-T-N-V-N-C-S-S-P-E-R-H-T-R- (SEQ ID NO: 2), of themature, full-length rKGF.

Another one of the objects of the present invention is to provide a KGFfragment, as above, that has decreased cytotoxicity as compared to themature, full-length rKGF.

Still another one of the objects of the present invention is to providea conjugate that comprises the KGF fragment described above and a toxinmolecule. The toxin molecule can be one of a ricin A molecule, adiphtheria toxin molecule, or a saporin molecule.

Yet another one of the objects of the present invention is to provide atherapeutic composition that contains the KGF fragment as describedabove and a pharmaceutically acceptable carrier, for example, onesuitable for topical application to human skin.

Still another one of the objects of the present invention is to providea DNA molecule that is composed of a nucleotide sequence that encodesthe KGF fragment described above.

Yet another one of the objects of the present invention is to provide anexpression vector that contains the DNA molecule that encodes the KGFfragment above and a regulatory sequence for expression of the DNAmolecule. The expression vector can be, for example, a yeast, abacterial, a mammalian or a baculovirus expression vector.

Yet another one of the objects of the present invention is to provide ahost cell transformed with the expression vector described above. Thehost cell can be, for example, a prokaryote such as a bacterial cell, ora eukaryote such as a yeast cell, a mammalian cell, or an insect cell.

Yet another one of the objects of the present invention is to provide amethod of producing the KGF fragment by culturing the transformed hostcell as described above and isolating the KGF fragment from the culture.

Still another one of the objects of the present invention is to providea method of stimulating epithelial cell growth by applying the KGFfragment to an area in which epithelial cell growth is desired andallowing the cells to grow.

Still another one of the objects of the present invention is to providea method for wound healing by applying the therapeutic compositiondescribed above to an area of a wound to be treated and allowing thewound to heal.

Still another one of the objects of the present invention is to providea method of treating a hyperproliferative disease of the epidermis byapplying the conjugate described above to an area to be treated.

Further objects, features, and advantages of the present invention wouldbe apparent to a person of ordinary skill in the art and need not beenumerated here.

SUMMARY OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of the mature, full-length, humanKGF polypeptide, beginning with the N-terminus and containing 163 aminoacid residues, having a single consensus sequence for glycosylation atamino acid residues 14-16. The amino acid sequence of the mature KGFpolypeptide corresponds to amino acids nos. 32 to 194 of SEQ ID NO: 1.

FIG. 2 shows the pAcC13 expression vector into which the 163 amino acidsequence of KGF has been inserted. The polylinker and flanking sequencesshown in FIG. 2 are referred as SEQ ID NO: 19 in the Sequence Listing.

FIG. 3 illustrates the protein elution profile (A) and bioactivityprofile (B) of the rKGF obtained from a Mono S HR5/5 cation exchangeFPLC column.

FIG. 4 compares the biological activity of the long, i.e., full-lengthrKGF, and the short, i.e., rKGF_(des1-23), form of KGF versus aFGF onthe cells of the Balb/Mk cell line. The figure legend for FIG. 4 is asfollows: ◯=KGF_(des1-23), =KGF₁₆₃, and □=aFGF.

FIG. 5 compares the biological activity of the long, i.e., full-lengthrKGF, and the short, i.e., rKGF_(des1-23), form of KGF versus bFGF onvascular endothelial cells derived from large vessels (A:ABAE cells) orcapillary cells (B:ACE cells).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It has been surprisingly discovered that a truncated, unglycosylated KGFhaving a deletion spanning the first 23 N-terminal amino acid residuesof mature recombinant KGF ("rKGF"), herein designated the KGF fragmentor KGF_(des1-23), possesses a much greater biological activity anddecreased cytotoxicity on epithelial cells as compared with the mature,full-length rKGF. Generally, the KGF fragment of the present inventionretains the specificity of KGF for stimulation of epithelial cellproliferation.

The following definitions are incorporated herein and are provided for abetter understanding of the present invention.

Definitions

As used herein, the term "keratinocyte growth factor" or "KGF" refers toa member of a group of structurally distinct proteins known as FGFs thatdisplay varying degrees of sequence homology, suggesting that they areencoded by a related family of genes. The FGFs share common receptorsites on cell surfaces. KGF, for example, can bind to FGFR-3.

"Mature, full-length KGF" or "long form of KGF" or "KGF₁₆₃," as usedherein refers to the mature polypeptide that contains 163 amino acidresidues, as shown in FIG. 1, and represented by amino acid residues 32to 194 in SEQ ID NO: 1.

As used herein, "the KGF fragment" or "short form of KGF" or"KGF_(des1-23) " refers to a polypeptide that is a truncated version ofKGF₁₆₃, lacking the first 23 amino acid residues at the N-terminus ofKGF₁₆₃. The sequence of the 23 deleted amino acid residues is shown inSEQ ID NO: 2. The properties of the KGF fragment include (i) itsbiological activity such as at least a 2-fold, preferably, a 7-fold and,more preferably, a 7-10 fold increase in stimulation of epithelial cellproliferation as compared to the rKGF₁₆₃ molecule, and (ii) its abilityto bind to FGFR-3. The biological activity of the KGF fragment can bemeasured, for example, by the Balb/Mk cell proliferation assay,described in Example 5, section C.

An "analog of KGF₁₆₃ " or "analog of the KGF fragment" herein refers toamino acid insertions, deletions, or substitutions in the relevantmolecule that do not substantially affect its properties. TheKGF_(des1-23) analog herein retains at least the 2-fold increase,preferably, the 7-fold increase, more preferably, the 7-10 fold increasein mitogenic activity as compared to that of rKGF₁₆₃. For example, theanalog herein can include conservative amino acid substitutions in therKGF_(des1-23) molecule.

"Conservative amino acid substitutions" herein are, for example, thosethat take place within a family of amino acids that are related in theirside chains. The families of amino acids include (1) acidic: asparticacid, glutamic acid; (2) basic: lysine, arginine, histidine; (3)non-polar: alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan; and (4) uncharged polar: glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine,tryptophan, and tyrosine are sometimes classified jointly as a family ofaromatic amino acids. In particular, it is generally accepted that anisolated replacement of a leucine with an isoleucine or valine, or anaspartic acid with a glutamic acid, or a threonine with a serine, or asimilar conservative substitutions of an amino acid with a structurallyrelated amino acid, in an area outside of the polypeptide's active sitewill not have a major effect on the properties of the polypeptide.

The term "recombinant" as used herein in relation to a polynucleotideintends a polynucleotide of semisynthetic, or synthetic origin, orencoded by cDNA or genomic DNA ("gDNA") such that (1) it is notassociated with all or a portion of a polynucleotide with which it isassociated in nature, (2) is linked to a polynucleotide other than thatto which it is linked in nature, or (3) does not occur in nature.

A "expression vector" is a polynucleotide that is operable in a desiredhost cell and capable of causing the production of the KGF fragment.Examples of expression vectors are plasmids, integrating vectors, etc.

A "regulatory sequence" refers to a polynucleotide sequence that isnecessary for regulation of expression of a coding sequence to which thepolynucleotide sequence is operably linked. The nature of suchregulatory sequences differs depending upon the host organism. Inprokaryotes, such regulatory sequences generally include, for example, apromoter, and/or a transcription termination sequence. In eukaryotes,generally, such regulatory sequences include, for example, a promoterand/or a transcription termination sequence. The term "regulatorysequence" may also include additional components the presence of whichare advantageous, for example, a secretory leader sequence for secretionof the polypeptide attached thereto.

"Operably linked" refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. A regulatory sequence is "operably linked" to a codingsequence when it is joined in such a way that expression of the codingsequence is achieved under conditions compatible with the regulatorysequence.

A "coding sequence" is a polynucleotide sequence that is translated intoa polypeptide, usually via mRNA, when placed under the control of anappropriate regulatory sequence. The boundaries of the coding sequenceare generally determined by a translation start codon at its 5'-terminusand a translation stop codon at its 3'-terminus. A coding sequence caninclude, but is not limited to, cDNA, and recombinant polynucleotidesequences.

An "open reading frame" (ORF) is a region of a polynucleotide sequencethat encodes a polypeptide. This region may encode a precursor form of amature polypeptide or just the polypeptide.

"PCR" refers to the techniques of the polymerase chain reaction asdescribed in Saiki, et al., Nature 324:163 (1986); and Scharf et al.,Science 233: 1076-1078 (1986); U.S. Pat. No. 4,683,195; and U.S. Pat.No. 4,683,202. As used herein, x is "heterologous" with respect to y ifx is not naturally associated with y or x is not associated with y inthe same manner as is found in nature.

"Homology" refers to the degree of sequence identity between x and y.Typically, the sequence identity between x and y will be at least 50%,usually, the sequence identity will be no less than 60%; more typically,the sequence identity will be no less than 75%; preferably no less than80%; and even more preferably at least 90%. Most preferably, thesequence identity between x and y will be at least 95%, even morepreferably at least 98%, even more preferably at least 99%.

As used herein, the term "polypeptide" refers to a polymer of aminoacids and does not refer to a specific length of the product. Thus,peptides, oligopeptides, and proteins are included within the definitionof polypeptide. This term also does not exclude post-expressionmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like. Included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids),polypeptides with substituted linkages, as well as other modificationsknown in the art, both naturally occurring and non-naturally occurring.

As used herein, "terminators" are regulatory sequences, such aspolyadenylation and transcription termination sequences, located 3' ordownstream of the stop codon of the coding sequences.

"Recombinant host cells," "host cells," "cells," "cell cultures," andother such terms denote, for example, microorganisms, insect cells, andmammalian cells, that can be or have been used as recipients forintroduction of recombinant vector or other transfer DNA, and includethe progeny of the cell that has been transformed. Such progeny includesthose that may not necessarily be identical in morphology or in genomicor total DNA complement as the original parent that may be produced as aresult of natural, accidental, or deliberate mutation. Examples ofmammalian host cells include Chinese hamster ovary ("CHO") and monkeykidney ("COS") cells.

As used herein, the term "microorganism" includes prokaryotic andeukaryotic microbial species such as bacteria and fungi, the latterincluding yeast and filamentous fungi.

"Transformation," as used herein, refers to the transfer of an exogenouspolynucleotide into a host cell, irrespective of the method used for thetransfer, which can be, for example, by infection, direct uptake,transduction, F-mating, microinjection or electroporation. The exogenouspolynucleotide may be maintained as a non-integrated vector, forexample, a plasmid, or alternatively, may be integrated into the hostgenome.

"Purified" and "isolated" in reference to a polypeptide or a nucleotidesequence means that the indicated molecule is present in substantialabsence of other biological macromolecules of the same species or type.The term "purified" as used herein means at least 75% by weight;preferably, at least 85% by weight, more preferably, at least 95% byweight and, most preferably, at least 98% by weight, of biologicalmacromolecules of the same type are present, but water, buffers, andother small molecules, especially molecules having a molecular weight ofless than 1000, can be present as well.

By "pharmaceutically acceptable carrier," is meant any carrier that isused by persons in the art for administration into a human that does notitself induce any undesirable side effects such as the production ofantibodies, fever, etc. Suitable carriers are typically large, slowlymetabolized macromolecules that can be a protein, a polysaccharide, apolylactic acid, a polyglycolic acid, a polymeric amino acid, amino acidcopolymers or an inactive virus particle. Such carriers are well knownto those of ordinary skill in the art. Preferably the carrier isthyroglobulin.

A "therapeutic composition" herein contains one or more pharmaceuticallyacceptable carriers, and one or more additional component such as water,saline, glycerol, or ethanol. Additionally, auxiliary substances, suchas wetting or emulsifying agents, pH buffering substances, and the like,may be present in such compositions.

By "a therapeutically effective amount," as used herein refers to thatamount that is effective for production of a desired result. This amountvaries depending upon the health and physical condition of theindividual to be treated, the capacity of the individual's immune systemto synthesize antibodies, the degree of protection desired, theformulation, the attending physician's assessment of the medicalsituation, and other relevant factors. It is expected that the amountwill fall in a relatively broad range that can be determined throughroutine trials.

Accordingly, a preferred embodiment of the present invention is a novelunglycosylated KGF fragment, KGF_(des1-23), unaccompanied by impuritieswhich normally accompany the native molecule when it is produced invivo. This fragment has an apparent molecular weight of about 18kilo-Daltons ("kD") based upon its migration in Sodium Dodecyl SulfatePolyacrylamide Gel Electrophoresis ("SDS/PAGE") as a single band(results not shown). The specific activity of purified KGF_(des1-23) onBalb/Mk cells is measured by its ED₅₀ value, as defined by theconcentration that causes half maximal stimulation of cellproliferation. The ED₅₀ of the KGF fragment herein is found to averageabout 40 pg/ml. The bioactivity of the KGF fragment herein will be atleast about 2-fold, preferably, about 7-fold and, more preferably, about7-10 fold greater than that of the full-length rKGF protein or that ofaFGF, when compared in a cell proliferation assay, and 100-fold greaterthan when rKGF₁₆₃ is bioassayed, using initiation of DNA synthesis inBalb/Mk cells maintained in a chemically defined medium, as described inPCT Patent Application, No. WO 90/08771.

In a preferred embodiment of the present invention, KGF_(des-1-23) isproduced by recombinant DNA technology, particularly in the case oflarge-scale commercial production. A recombinant DNA molecule and anexpression vector comprising KGF_(des1-23) in accordance with thepresent invention can be made and expressed by conventional geneexpression technology using methods well-known in the art, as discussedin more detail below.

The present invention also includes analogs of the rKGF fragment thatretain the epithelial cell specificity and the at least 2-fold increasein mitogenic activity of rKGF_(des1-23), as compared to the activity ofrKGF₁₆₃. In a preferred embodiment, the analog retains the 7-fold, morepreferably, the 7-10 fold and, most preferably, the 10-fold increase inmitogenic activity of rKGF_(des1-23). Analogs of rKGF_(des1-23) includepost-translationally modified versions of rKGF_(des1-23), for example,those generated by glycosylations, acetylations, or phosphorylationsthereof. Analogs of rKGF_(des1-23) can be also made by conventionaltechniques of amino acid substitution, deletion, or addition, forexample, by site-directed mutagenesis. Thus, all references toembodiments of the present invention as it relates to rKGF₁₋₂₃ applyequally to analogs thereof.

In one embodiment of the present invention, the KGF fragment can be madeby isolating native, mature KGF from cells producing the same anddeleting the first 23 N-terminal amino acid residues therefrom. Suchdeletion can be performed by any conventional techniques known in theart.

In an alternative embodiment, the KGF fragment can be made by isolatingthe coding sequence of native KGF₁₆₃, deleting the codons that encodethe first 23 N-terminal amino acid residues, inserting the modifiedcoding sequence into an expression vector, and transforming host cellswith the expression vector to produce the recombinant KGF_(des1-23).

In a further embodiment of the present invention, the KGF fragment canbe made by isolating the coding sequence of KGF₁₆₃ from cells known toproduce KGF, inserting the coding sequence of KGF₁₆₃ into a baculovirusexpression vector, transforming a host insect cell with the baculovirusexpression vector, and harvesting and isolating the approximately 18 kDmolecular species from the transformed insect cell culture thatpossesses increased KGF activity, using conventional separatorytechniques.

In another embodiment of the present invention, an expression vectorcontaining the KGF_(des1-23) coding sequence can be produced by operablylinking the KGF_(des1-23) to one or more regulatory sequences such thatthe resulting vector is operable in a desired host.

In a further embodiment of the present invention, the coding sequence ofKGF_(des1-23) can be obtained by conventional techniques, including theisolation of the coding sequence of KGF₁₆₃ from a cDNA library known tocontain such, and deleting therefrom the sequence encoding the first 23N-terminal amino acid residues. Deletion of the coding sequence of theN-terminal amino acids can be accomplished in vivo or in vitro. Theformer can be achieved, for example, by expression of the KGF₁₆₃ codingsequence in a baculovirus/insect cell expression system. The latter canbe achieved by known PCR techniques using primers that exclude theN-terminal sequences.

In a further embodiment of the present invention, the DNA or vectorcomprising the coding sequence of KGF_(des1-23) can be expressed in aprokaryotic or eukaryotic expression system, in particular, a bacterial,mammalian, yeast, or insect cell expression system. In a preferredembodiment, a bacterial or yeast cell expression system may be ideal forproduction of the KGF_(des1-23) fragment. The yeast cell can be, forexample, Saccharomyces cerevisiae.

In another embodiment of the present invention, the KGF fragment can beexpressed as a fusion protein by linking, in the correct frame andorientation, the 5' end of the KGF_(des1-23) coding sequence to thecoding sequence of another molecule that facilitates eitherintracellular or extracellular production of the rKGF_(des1-23). Thecoding sequence of such other molecules can be, for example, at least aportion of the prepro α-factor leader sequence, for extracellularexpression; the superoxide dismutase ("SOD") gene sequence, or theubiquitin gene sequence, for intracellular expression in yeast cells.

In yet another embodiment of the present invention, the rKGF_(des1-23)polypeptide can be conjugated to other molecules suitable for itsintended use. For example, the KGF_(des1-23) polypeptide can beconjugated to a toxin molecule, such as ricin A, diphtheria toxin, orsaporin for destruction of its target cell, i.e., epithelial cells,particularly, keratinocytes.

In a further embodiment, the KGF_(des1-23) polypeptide or a conjugatethereof can be mixed with a pharmaceutically acceptable carrier toproduce a therapeutic composition that can be administered fortherapeutic purposes, for example, for wound healing, and for treatmentof hyperproliferative diseases of the skin and tumors, such as psoriasisand basal cell carcinoma.

The KGF fragment of the present invention can be used for identificationof receptor recognition sites as well as for the design of peptideagonists or antagonists. Moreover, in view of the unique specificity ofKGF for keratinocytes, its inability to induce the proliferation ofvascular endothelial cells or fibroblasts, and its lack of cytotoxicity,KGF_(des1-23) should be a preferred agent of choice for wound healingapplications, particularly where there is a desire to promotere-epithelialization of the skin. KGF_(des1-23) should also beparticularly useful in corneal epithelial repair. Other applications ofKGF₁₋₂₃ utilize its specificity for epithelial cells found in thegastrointestinal tract.

The choice to select the KGF fragment herein over other growth factorssuch as epidermal growth factor ("EGF"), platelet-derived growth factor("PDGF"), and other FGFs for skin repair is within the skill of a personin the art. These other growth factors, for example, induce fibroplasiaand angiogenesis, in addition to stimulating, either directly orindirectly, keratinocyte proliferation. In skin repair, such additionalactivities could produce undesirable side effects such as scarring. Incorneal repair involving either a wound or surgery, the use of thesefactors could induce blood vessel invasion into the cornea, and resultin corneal opacity or edema. KGF, on the other hand, has a uniquespecificity for keratinocytes and does not induce the proliferation ofvascular endothelial cells or fibroblasts and, therefore, would be theagent of choice for these particular wound healing applications.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature, includingSambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd ed. (ColdSpring Harbor Laboratory Press, 1989); DNA CLONING, Vol. I and II, D. NGlover ed. (IRL Press, 1985); OLIGONUCLEOTIDE SYNTHESIS, M. J. Gait ed.(IRL Press, 1984); NUCLEIC ACID HYBRIDIZATION, B. D. Hames & S. J.Higgins eds. (IRL Press, 1984); TRANSCRIPTION AND TRANSLATION, B. D.Hames & S. J. Higgins eds., (IRL Press, 1984); ANIMAL CELL CULTURE, R.I. Freshney ed. (IRL Press, 1986); IMMOBILIZED CELLS AND ENZYMES, K.Mosbach (IRL Press, 1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULARCLONING, Wiley (1984); the series, METHODS IN ENZYMOLOGY, AcademicPress, Inc.; GENE TRANSFER VECTORS FOR MAMMALIAN CELLS, J. H. Miller andM. P. Calos eds. (Cold Spring Harbor Laboratory, 1987); METHODS INENZYMOLOGY, Vol. 154 and 155, Wu and Grossman, eds., and Wu, ed.,respectively (Academic Press, 1987), IMMUNOCHEMICAL METHODS IN CELL ANDMOLECULAR BIOLOGY, R. J. Mayer and J. H. Walker, eds. (Academic PressLondon, Harcourt Brace U.S., 1987), PROTEIN PURIFICATION: PRINCIPLES ANDPRACTICE, 2nd ed. (Springer-Verlag, N.Y. (1987), and HANDBOOK OFEXPERIMENTAL IMMUNOLOGY, Vol. I-IV, D. M. Weir et al., (BlackwellScientific Publications, 1986); Kitts et al., Biotechniques 14:810-817(1993); Munemitsu et al., Mol. and Cell. Biol. 10:5977-5982 (1990).

Standard abbreviations for nucleotides and amino acids are used in FIG.1 and elsewhere in this specification. All publications, patents, andpatent applications cited herein are incorporated by reference.

It has been unexpectedly found that when KGF vector is expressed ininsect cells, Spodoptera frugiperda ("SF9") by infection of arecombinant baculovirus, Autographa californica, containing the cDNAcoding for the mature form of KGF (163 amino acids), they were capableof producing a KGF fragment, a truncated form of KGF that lacks thefirst 23 amino acid residues of the native KGF N-terminal domain thatcontains a single glycosylation site. This truncated and unglycosylatedKGF fragment designated herein as KGF_(des1-23) or KGF₁₄₀, in contrastto the native long form identified as KGF₁₆₃, has a 7- to 10-foldincreased potency on target cells. The target cell specificity ofKGF_(des1-23) is unchanged. Additionally, at high concentrations of theKGF fragment, no toxic effect on keratinocytes is observed in contrastto that observed for KGF₁₆₃. These observations suggest that, contraryto what was previously proposed in PCT Application, Publication No. WO90/08771, the target cell specificity of KGF does not reside in itsN-terminal domain. Furthermore, the present invention shows that anN-terminally truncated version of KGF in fact represents an improved KGFversion with higher biological activity and decreased cytotoxicity fortherapeutic application.

Further, KGF_(des1-23) exhibits a pI more basic than that for KGF₁₆₃. Inone assay, the pI was approximately 9.9 for KGF_(des1-23) and 9.4 forKGF_(des1-23). KGF_(des1-23) also exhibits a higher affinity for heparinand for the Mono S resin.

Recombinant KGF_(des1-23), in accordance with the present invention, canbe made by well-known recombinant techniques. In this regard, theKGF_(des1-23) coding sequence is operably linked to one or moreregulatory sequences in the suitable vector in a proper reading frameand orientation. The vector is suitable when it can replicate or can bereplicated to express the recombinant protein in a particular host.Thus, the coding sequence of KGF_(des1-23) can be inserted, for example,into a yeast expression vector for expression in yeast cells, abacterial expression vector for expression in bacterial cells, amammalian vector for expression in mammalian cells.

In a preferred embodiment, the coding sequence of KGF_(des1-23) forexpression purposes herein is a complementary DNA ("cDNA") moleculeencoding KGF_(des1-23). The KGF_(des1-23) cDNA can be made by knownrecombinant techniques, such as isolating total cellular RNA from a hostcell known to express KGF, isolating poly A⁺ RNA by running the totalcellular RNA through an oligo-dT column and eluting the poly A⁺ RNAtherefrom, constructing a cDNA library, using reverse transcriptase,based on the poly A⁺ RNA isolated, which contains mRNA, and selectingthe cDNA clones that contain the KGF_(des1-23) coding sequence by use oflabeled oligonucleotide probes constructed on the basis of the knownamino acid sequence of KGF_(des1-23).

A regulatory sequence that can be linked to the KGF_(des1-23) codingsequence in the expression vector herein is a promoter that is operablein the host cell in which the recombinant KGF_(des1-23) is to beexpressed. Optionally, other regulatory sequences can be used herein,such as one or more of an enhancer sequence, an intron with functionalsplice donor and acceptance sites, a signal sequence for directingsecretion of the recombinant KGF_(des1-23), a polyadenylation sequence,other transcription terminator sequences, and a sequence homologous tothe host cell genome. Other sequences, such as an origin of replication,can be added to the vector as well to optimize expression of the desiredproduct. Further, a selectable marker can be present in the expressionvector for selection of the presence thereof in the transformed hostcells.

The regulatory sequences can be derived from various sources. Forexample, one or more of them can be associated with a native KGF codingsequence, or derived from or homologous with the host cell in which thesequence is to be expressed, or derived from a microbial, for example,bacterial or yeast source, or hybrids thereof. For use herein, theKGF_(des1-23) coding sequence is, preferably, operably linked downstreamof the promoter sequence and upstream of the terminator sequence.

The various components of the expression vector can be linked togetherdirectly or, preferably, via linkers that constitute sites ofrecognition by restriction enzymes. In one embodiment of the presentinvention, a promoter sequence herein is linked directly with theKGF_(des1-23) coding sequence, in which case the first amino acid at theN-terminal of the recombinant KGF_(des1-23) protein would be methionine,which is encoded by the ATG start codon. The methionine residue at theN-terminal can be optionally cleaved from the recombinant protein byconventional techniques, for example, in vitro incubation with cyanogenbromide provided no other methionine residues are present in thefragment.

Any promoter that would allow expression of the KGF fragment in adesired host can be used in the present invention. Yeast promotersequences that are associated with polynucleotide sequences encodingenzymes in the fermentative metabolic pathway are particularly useful inthe present invention. Examples of these enzymes include alcoholdehydrogenase ("ADH"), as described in European Patent Application,Publication No. 284 044, enolase, glucokinase, glucose-6-phosphateisomerase, glyceraldehyde-3-phosphate-dehydrogenase ("GAP" or "GAPDH"),hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvatekinase ("PyK"), as described in European Patent Application, PublicationNo. 329 203. In addition, the yeast PHO5 gene, encoding acidphosphatase, as described in Myanohara et al., Proc. Natl. Acad. Sci.USA 80:1 (1983), is also useful as a promoter sequence herein.

Other yeast promoters that are suitable herein include, for example,those described in Cohen et al., Proc. Natl. Acad. Sci. USA 77:1078(1980); Henikoff et al., Nature 283:835 (1981); Hollenberg et al., Curr.Topics Microbiol. Immunol. 96:119 (1981); Hollenberg et al., "TheExpression of Bacterial Antibiotic Resistance Genes in the YeastSaccharomyces cerevisiae," in PLASMIDS OF MEDICAL, ENVIRONMENTAL ANDCOMMERCIAL IMPORTANCE, K. N. Timmis and A. Puhler, eds. (Amsterdam/NewYork 1979); Mercerau-Puigalon et al., Gene 11:163 (1980); Panthier etal., Curr. Genet. 2:109 (1980).

Prokaryotic promoter sequences, optionally containing operator portions,that can be used herein include β-lactamase (penicillinase) and lactosepromoter systems, as described in Chang et al., Nature 198:1056 (1977),tryptophan promoter system, as described in Goeddel et al., NucleicAcids Res. 8:4057 (1980), and the λ (lambda)-derived P_(L) promoter.

Preferred mammalian promoter sequences that can be used herein are thosefrom mammalian viruses that are highly expressed and that have a broadhost range. Examples include the SV40 early promoter, theCytomegalovirus ("CMV") immediate early promoter mouse mammary tumorvirus long terminal repeat ("LTR") promoter, adenovirus major latepromoter (Ad MLP), and Herpes Simplex Virus ("HSV") promoter. Inaddition, promoter sequences derived from non-viral genes, such as themurine metallothionein gene, are also useful herein. These promoters canfurther be either constitutive or regulated, such as those that can beinduced with glucocorticoids in hormone-responsive cells.

Promoters that can be used for insect cell expression in the presentinvention include the baculovirus polyhedron hybrid promoter and the p10promoter.

Further, promoters for use in the present invention can be synthetichybrid promoters that do not occur in nature ("non-natural promoters"),but contain a regulatory region linked with a heterologous expressioninitiation region. For example, the UAS sequence of one yeast promotermay be joined with the transcription activation region of another yeastpromoter, creating a synthetic hybrid promoter. Examples of suchpromoters include the ADH regulatory sequence linked to the GAPtranscription activation region, as described in U.S. Pat. Nos.4,876,197 and 4,880,734. Other examples include those containingregulatory sequences that are associated with the coding sequencesencoding ADH2, GAL4, GAL10, or PHO5, combined with the transcriptionalactivation region of a glycolytic enzyme, such as GAP or PyK, asdescribed in U.S. Pat. Nos.: 4,876,197 and 4,880,734.

Bacterial hybrid promoters include, for example, the tac promoter, asdescribed in De Boer et al. (1983), that is derived from sequences ofthe trp and lac UV5 promoters. A functional non-natural promoter for useherein may also be a synthetic promoter that is based on a consensussequence of different promoters.

In another embodiment of the present invention, an enhancer element canbe combined with a promoter sequence. Such enhancers not only amplifybut also can regulate expression of the rKGF_(des1-23) polypeptide.Suitable enhancer elements for use in mammalian expression systems are,for example, those derived from viruses that have a broad host range,such as the SV40 early gene enhancer, as described in Dijkema et al.,EMBO J. 4:761 (1985), the enhancer/promoters derived from the LTR of theRous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad.Sci. 79:6777 (1982), and from human cytomegalovirus, as described inBoshart et al., Cell 41:521 (1985). Additionally, other suitableenhancers include those that can be incorporated into promoter sequencesthat will become active only in the presence of an inducer, such as ahormone, a metal ion, or an enzyme substrate, as described inSassone-Corsi and Borelli, Trends Genet. 2:215 (1986); and Maniatis etal., Science 236:1237 (1987).

For expression in yeast cells, a yeast promoter is preferably used thatcontains an upstream activator sequence ("UAS") that permits regulatedexpression of the recombinant KGF_(des1-23). Regulated expression hereinmay be either positive or negative, thereby either enhancing or reducingtranscription. In an alternative embodiment, a UAS can be absent, inwhich instance, constitutive expression of KGF_(des1-23) would occur.

In another embodiment of the present invention, a transcriptiontermination sequence is placed 3' to the translation stop codon of theKGF_(des1-23) coding sequence. Thus, the terminator sequence, togetherwith the promoter, flank the KGF_(des1-23) coding sequence. Examples oftranscription terminator sequences are the yeast-recognized sequencesassociated with the yeast glycolytic enzymes and those derived fromSV40.

In one embodiment of the present invention, the recombinantKGF_(des1-23) can be made to be secreted from the host cell into thegrowth media by creating chimeric DNA molecules that encode a fusionprotein comprised of a leader sequence that contains a secretory signalsequence for secretion of KGF_(des1-23). For the purpose of the presentinvention, the signal sequences that are suitable for use herein are,for example, those derived from genes for secreted endogenous host cellproteins, such as for the yeast expression system, the yeast invertasegene, as described in European Patent No. 012 873 and Japanese PatentApplication, Publication No. 62,096,086, the A-factor gene, as describedin U.S. Pat. No. 4,588,684, the prepro α-factor gene, as described inU.S. Pat. No. 4,870,008, and the interferon gene, as described inEuropean Patent No. 060 057.

In a preferred embodiment of the present invention, a truncated yeastα-factor leader sequence can be used. The truncated α-factor leadersequence contains at least a portion of the signal "pre" sequence, and aportion of the "pro" sequence that contains a glycosylation site.Typically, the α-factor leader sequences that can be employed hereininclude about 25 to about 50 amino acid residues of the N-terminalthereof, as described in U.S. Pat. Nos. 4,546,083 and 4,870,008, and incopending U.S. patent application Ser. No. 07/864,206 and EuropeanPatent Application, Publication No. 324 274. Also suitable for useherein are hybrid α-factor leaders made with a "pre" sequence of a firstyeast signal sequence, and a "pro" region from a second yeast α-factor,as described in PCT Application, Publication No. WO 89/02463. In oneembodiment of the present invention, the signal sequence contains aprocessing site 5' of the coding sequence of KGF_(des1-23), to allowcleavage thereof, either in vivo or in vitro. For mammalian expression,an example of a suitable leader sequence is the adenovirus tripartiteleader that provides for secretion of an operably linked KGF_(des1-23)protein in mammalian cells.

Prokaryotic leader sequences for directing the secretion of recombinantKGF_(des1-23) that are suitable for use herein are those known in theart. For example, such leader sequences include those disclosed in U.S.Pat. No. 4,336,336, relating to "Fused Gene and Method of Making andUsing the Same," to T. J. Silhavy et al., issued on Jun. 22, 1982, andU.S. Pat. No. 5,010,015, relating to "Recombinant DNA Molecules andMethod for Protein Production," to Palva, I., issued on Apr. 23, 1991.

In another embodiment of the present invention, the expression vectorcan contain an origin of replication such that it can be maintained as areplicon, capable of autonomous replication and stable maintenance in ahost. Such an origin of replication includes those that enable anexpression vector to be reproduced at a high copy number in the presenceof the appropriate proteins within the cell, for example, the 2μ andautonomously replicating sequences that are effective in yeast, and theorigin of replication of the SV40 vital T-antigen, that is effective inCOS-7 cells.

Mammalian replication systems that are suitable for the presentinvention include those derived from animal viruses that requiretrans-acting factors to replicate. For example, the replication systemof papovaviruses, such as SV40, as described in Gluzman, Cell 23:175(1981), or polyomavirus that replicate to extremely high copy number inthe presence of the appropriate vital T antigen. Additional examplesinclude those derived from bovine papillomavirus and Epstein-Barr virus.

Additionally, the expression vector herein can have more than onereplication system, thus, allowing it to be maintained, for example, inmammalian cells for expression and in a procaryotic host for cloning andamplification. Examples of such mammalian-bacteria shuttle vectorsinclude pMT2, as described in Kaufman et al., Mol. Cell. Biol. 9:946(1989) and pHEBO, as described in Shimizu et al., Mol. Cell. Biol.6:1074 (1986). Examples of yeast-bacteria shuttle vectors include YEp24,as described in Botstein et al., Gene 8:17-24 (1979); pC1/1, asdescribed in Brake et al., Proc. Natl. Acad. Sci USA 81:4642-4646(1984); and YRp17, as described in Stinchcomb et al., J. Mol. Biol.158:157 (1982).

The replicon herein may be either a high or low copy number plasmid. Ahigh copy number plasmid will generally have a copy number ranging fromabout 5 to about 200, and typically about 10 to about 150. A hostcontaining a high copy number plasmid will preferably have at leastabout 10, and more preferably at least about 20. Either a high or lowcopy number vector can be used herein, depending upon the effect of thevector and the recombinant KGF_(des1-23) on the host. See e.g., Brake etal. (loc. cit. ).

In another embodiment of the present invention, the expression vectorcan be made to integrate into the host cell genome as an integratingvector. The integrating vector herein contains at least onepolynucleotide sequence that is homologous to the host cell genome thatallows the vector to integrate. Preferably, the host cell contains twohomologous sequences flanking the KGF_(des1-23) coding sequence, asdescribed in, for example, European Patent Application, Publication No.127 328, relating to integrating vectors constructed with DNA fromvarious Bacillus strains integrated into the Bacillus chromosome.Integrating vectors may also be comprised of bacteriophage or transposonsequences.

The homologous sequences, however, need not be linked to the expressionvector. For example, an expression vector can be used that can integrateinto the CHO genome via an unattached dihydrofolate reductase gene. In apreferred embodiment, the homologous sequences flank the KGF_(des1-23)coding sequence in the vector. Particularly useful homologous yeastgenome sequences for the present invention are, for example, thosedisclosed in PCT Application, Publication No. WO 90/01800, and the his4gene sequences, as described in Genbank, accession no. J01331.

In another embodiment of the present invention, one or more selectablemarkers can be attached to other components in the expression vector toallow for the selection of the host cells that have been transformed.Selectable markers that can be expressed in a host cell include genesthat can render the host cell resistant to drugs such as tunicamycin,G418, ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin),and tetracycline, as described in Davies et al., Ann. Rev. Microbiol.32:469 (1978). Selectable markers herein also include biosyntheticgenes, such as those in the histidine, tryptophan, and leucinebiosynthetic pathways, such as ade2, his4, leu2, trp1. Thus, forexample, when a leu⁻ host cell is used as recipient in transformationwith an expression vector, and leucine is absent from the media, forexample, only the cells that carry a plasmid with a leu⁺ gene willsurvive.

In addition, selectable markers that provide the host cells with theability to grow in the presence of toxic compounds, such as metal, isalso suitable herein; for instance, the presence of cup1 allows yeaststo grow in the presence of copper ions, as described in Butt et al.,Microbiol. Rev. 51:351 (1987).

The method for construction of an expression vector for transformationof insect cells for expression of recombinant KGF_(des1-23) herein isslightly different than that generally applicable to the construction ofa bacterial expression vector, a yeast expression vector, or a mammalianexpression vector. In an embodiment of the present invention, abaculovirus vector is constructed in accordance with techniques that areknown in the art, for example, as described in Kitts et al.,BioTechniques 14:810-817 (1993), Smith et al., Mol. Cell. Biol. 3:2156(1983), and Luckow and Summer, Virol. 17:31 (1989). In one embodiment ofthe present invention, a baculovirus expression vector is constructedsubstantially in accordance to Summers and Smith, Texas AgriculturalExperiment Station Bulletin No. 1555 (1987). Moreover, materials andmethods for baculovirus/insect cell expression systems are commerciallyavailable in kit form, for example, the MaxBac® kit from Invitrogen (SanDiego, Calif.).

Briefly, a KGF expression cassette including the KGF_(des1-23) codingsequence and optionally, a regulatory sequence, a selectable marker,etc., can be first constructed by insertion of the KGF₁₆₃ orKGF_(des1-23) coding sequence into a baculovirus transfer vector thatcontains at least an essential polynucleotide sequence, as described inmore detail below, and/or a polynucleotide sequence that is homologousto a portion of the baculovirus genome ("the baculovirus sequences") andis capable of homologous recombination therewith, for example, thepolyhedron gene. Insertion therein can be engineered at a restrictionenzyme site, as described in Miller et al., Bioassays 4:91 (1989). In apreferred embodiment, a KGF_(des1-23) coding sequence is positioneddownstream from the polyhedron promoter and flanked at both the 5' andthe 3' end by polyhedron-specific sequences.

The KGF₁₆₃ coding sequence can be obtained by known recombinanttechniques from cells that are known to express KGF activity. Thetransfer vector containing the KGF_(des1-23) coding sequence isco-transfected into host insect cells together with a mutant ofwild-type baculovirus, so as to form a recombinant baculovirus. Thismutant baculovirus lacks an essential polynucleotide sequence necessaryfor production of a functional recombinant virus. In this regard, afunctional virus is one that is capable of independent replication in ahost. A functional recombinant virus is obtained when the mutantbaculovirus and the transfer vector carrying the KGF expression cassetteboth infect a host insect cell and recombine therein.

Recombinant baculovirus can be identified by known methods. For example,the wild-type viruses produce a polyhedron protein at very high levelsin the nuclei of infected cells during a late stage of viral infection.Accumulated polyhedron protein forms occlusion bodies that also containembedded vital particles. These occlusion bodies, up to 15 μm in size,are highly refractile, giving them a bright shiny appearance that isreadily visualized under the light microscope. Cells infected withrecombinant viruses lack occlusion bodies. Recombinant virus andwild-type virus can, therefore, be distinguished by plating thetransfection supernatant or dilutions thereof onto a monolayer of insectcells by standard techniques. The plaques can then be screened under thelight microscope for the presence, indicative of wild-type virus, orabsence, indicative of recombinant virus, of occlusion bodies, asdescribed in Vol 2. of CURRENT PROTOCOLS IN MOLECULAR MICROBIOLOGY 16.8F. M. Ausubel et al. eds, Supp. 10, Green Publisher Associates and WileyInterscience (1990).

The functional recombinant baculovirus containing the KGF expressioncassette obtained in this manner is suitable for transfection into newhost insect cells for production of large quantities of the recombinantKGF_(des1-23). Recombinant KGF_(des1-23) produced in this manner can beseparated from KGF₁₆₃ based upon molecular weight differences, usingknown separatory techniques.

When making rather than purchasing a commercially availablebaculovirus/insect cell expression kit, the baculovirus transfer vectorherein can be made by linking different components together, such as apromoter, a secretory leader sequence, and a transcription terminationsequence. Alternatively, the transfer vector can be constructed suchthat a single copy of the KGF₁₆₃ coding sequence is operably linked toone or more regulatory elements, or multiple copies of the KGF_(des1-23)coding sequence are each operably linked to its own set of regulatoryelements. As a further alternative, multiple copies of the KGF_(des1-23)coding sequence can be regulated by the same set of regulatory elements.

The transfer vector for the present invention may optionally contain anorigin of replication so that it can be maintained as a replicon that iscapable of stable maintenance in a host, such as a bacterium, forcloning and amplification. In a preferred embodiment of the presentinvention, the transfer vector for introducing KGF_(des1-23) into AcNPVis pAc373. Other vectors, known to those of skill in the art, can alsobe used, including, for example, pVL985, which alters the polyhedrinstart codon from ATG to ATT, and which introduces a BamHI cloning site32 basepairs downstream from the ATT, as described in Luckow andSummers, Virol. 17:31 (1989). After the expression vector is made, thevector is used to transform host cells for expression of recombinantKGF_(des1-23). The host cells that are suitable for use herein includesprokaryotes and eukaryotes. The prokaryotes include Gram positive andGram negative bacteria. The eukaryotes include fungi, yeast, mammalian,and insect cells. The bacterial hosts include, for example,Campylobacter, Bacillus, Escherichia, Lactobacillus, Pseudomonas,Staphylococcus, and Streptococcus. Yeast hosts from the following generamay be utilized: Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, and Yarrowia. Mammalian cell linessuitable for use herein include, for example, many immortalized celllines available from the American Type Culture Collection (ATCC), suchas Chinese hamster ovary ("CHO") cells, HeLa cells, baby hamster kidney("BHK") cells, monkey kidney cells ("COS"), and human hepatocellularcarcinoma cells such as Hep G2. A number of insect cell hosts are alsosuitable for expression of the KGF fragment or analog, including, forexample, Aedes aegypti, Autographa californica, Bombyx mori, Drosophilamelanogaster, and Spodoptera frugiperda, as described in PCTApplication, Publication No. WO 89/046699; Carbonell et al., J. Virol.56:153 (1985); Wright Nature 321:718 (1986); Smith et al., Mol. Cell.Biol. 3:2156 (1983); and generally, Fraser, et al. in vitro Cell. Dev.Biol. 25:225 (1989).

Certain expression vectors, either extra-chromosomal replicons orintegrating vectors, have been developed for transformation into certainhosts. A person of ordinary skill in the art would be able to adopt oradapt one or more of these developed vectors for use herein. Forexample, expression vectors have been developed for the followingbacterial hosts that can be used herein: Bacillus subtilis, as describedPalva et al., Proc. Natl. Acad. Sci. USA 79:5582 (1982); European PatentNo. 036 259 U.S. Pat. No. 4,711,843 and European Patent Application,Publication No. 063 953; PCT Application, Publication No. WO 84/04541,U.S. Pat. No. 4,663,280; Escherichia coli, Shimatake et al., Nature292:128 (1981); Amann et al., Gene 40:183 (1985); Studier et al., J.Mol. Biol. 189:113 (1986); European Patent No. 036 776 and EuropeanPatent Application, Publication Nos. 136 829 and 136 907; Streptococcuscremoris, Powell et al., Appl. Environ. Microbiol. 54:655 (1988);Streptococcus lividans, Powell et al., Appl. Environ. Microbiol. 54:655(1988); Streptomyces lividans, U.S. Pat. No. 4,745,056.

Expression vectors that have been developed for yeasts that can be usedherein include, for example, Candida albicans as described in Kurtz, etal. Mol. Cell. Biol. 6:142 (1986); Candida maltosa in Kunze, et al. J.Basic Microbiol. 25:141 (1985); Hansenula polymorpha, in Gleeson, et al.J. Gen. Microbiol. 132:3459 (1986); Roggenkamp et al. Mol. Gen. Genet.202:302 (1986); Kluyveromyces fragilis, in Das, et al. J. Bacteriol.158:1165 (1984); Kluyveromyces lactis, in De Louvencourt et al. J.Bacteriol. 154:737 (1983); Van den Berg et al. Bio/Technology 8:135(1990); Pichia guillerimondii, in Kunze et al. J. Basic Microbiol.25:141 (1985); Pichia pastoris, in Cregg et al. Mol. Cell. Biol. 5:3376(1985); U.S. Pat. No. 4,837,148 and U.S. Pat. No. 4,929,555;Saccharomyces cerevisiae, in Hinnen et al. Proc. Natl. Acad. Sci. USA75:1929 (1978); Ito et al. J. Bacteriol. 153:163 (1983);Schizosaccharomyces pombe, in Beach and Nurse, Nature 300:706 (1981);and Yarrowia. lipolytica in Davidow, et al., Curr. Genet. 10:380-471(1985); Gaillardin et al., Curr. Genet. 10:49 (1985).

The transformation procedures suitable for use herein are those known inthe art and include, for example, first treating the bacterial cellswith CaCl₂ or other agents, such as divalent cations and DMSO, andincubating the treated cells with the KGF_(des1-23) coding sequence. DNAcan also be introduced into bacterial cells by electroporation. Theexact transformation procedure varies with the bacterial species to betransformed as described in, for example, Masson et al., FEMS Microbiol.Lett. 60:273 (1989); Palva et al., Proc. Natl. Acad. Sci. USA 79:5582(1982); European Patent Application, Pub. Nos. 036 259 and 063 953; PCTApplication, Publication No. WO84/04541, for Bacillus; Miller et al.,Proc. Natl. Acad. Sci. 85:856 (1988); Wang et al., J. Bacteriol. 172:949(1990), for Campylobacter; Cohen et al., Proc. Natl. Acad. Sci. 69:2110(1973); Dower et al., Nucleic Acids Res. 16:6127 (1988); Kushner "Animproved method for transformation of Escherichia coli withColE1-derived plasmids", in GENETIC ENGINEERING: PROCEEDINGS OF THEINTERNATIONAL SYMPOSIUM ON GENETIC ENGINEERING, H. W. Boyer and S.Nicosia, eds, Amsterdam and New York, Elsevier and North HollandBiomedical Press (1978); Mandel et al., J. Mol. Biol. 53:159 (1970);Taketo, Biochim. Biophys. Acta 949:318 (1988), for Escherichia; Chassyet al. , FEMS Microbiol. Lett. 44:173 (1987), for Lactobacillus; Fiedleret al., Anal. Biochem. 170:38 (1988), for Pseudomonas; Augustin et al.,FEMS Microbiol. Lett. 66:203 (1990), for Staphylococcus; Barany et al.,J. Bacteriol. 144:698 (1980); Harlander "Transformation of Streptococcuslactis by Electroporation", in STREPTOCOCCAL GENETICS, J. Ferretti andR. Curtiss III, eds (1987); Perry et al., Infec. Immun. 32:1295 (1981);Powell et al., Appl. Environ. Microbiol. 54:655 (1988); Somkuti et al.,Proc. 4th Evr. Cong. Biotechnology 1:412 (1987), for Streptococcus.

For yeast, the transformation procedures that can be used herein includeelectroporation, as described in "Guide to Yeast Genetics and MolecularBiology," Vol 194 METHODS IN ENZYMOLOGY, C. Guthrie and G. R. Pink,(Academic Press 1991). Other procedures include the transformation ofspheroplasts or the transformation of alkali cation-treated intactcells. Such procedures are described in, for example, Kurtz et al., Mol.Cell. Biol. 6:142 (1986); Kunze et al., J. Basic Microbiol. 25:141(1985), for Candida; Gleeson et al., J. Gen. Microbiol. 132:3459 (1986);Roggenkamp et al., Mol. Gen. Genet. 202:302, for Hansenula (1986); Daset al., J. Bacteriol. 158:1165 (1984); De Louvencourt et al., J.Bacteriol. 154:1165 (1983); Van den Berg et al., Bio/Technology 8:135(1990) for Kluyveromyces; Cregg et al., Mol. Cell. Biol. 5:3376 (1985);Kunze et al., J. Basic Microbiol. 25:141 (1985); U.S. Pat. No. 4,837,148and U.S. Pat. No. 4,929,555, for Pichia; Hinnen et al., Proc. Natl.Acad. Sci. USA 75;1929 (1978); Ito et al., J. Bacteriol. 153:163 (1983),for Saccharomyces; Beach and Nurse Nature 300:706 (1981), forSchizosaccharomyces; Davidow et al., Curr. Genet. 10:39 (1985);Gaillardin et al., Curr. Genet. 10:49 (1985), for Yarrowia.

For example, for mammalian cell systems, such methods includedextran-mediated transfection, calcium phosphate precipitation,polybrene-mediated transfection, protoplast fusion, electroporation,encapsulation of the KGF_(des1-23) polynucleotide in liposomes, anddirect microinjection of the DNA into nuclei.

Immunoassays and activity assays that are known in the art can beutilized herein to determine if the transformed host cells areexpressing the desired KGF fragment. For example, for detection ofintracellular production of KGF_(des1-23) by transformed host cells, animmunofluorescence assay can be performed on the transformed host cellswithout separating the KGF fragments from the cell membrane. In thisassay, the host cells are first fixed onto a solid support, such as amicroscope slide or microtiter well. Next, the fixed host cells areexposed to an anti-KGF antibody. Preferably, to increase the sensitivityof the assay, the fixed cells are exposed to a second antibody, that islabelled and binds to the anti-KGF antibody. For instance, the secondaryantibody may be labelled with an fluorescent marker. The host cellswhich express the KGF fragments will be fluorescently labelled can bevisualized under the microscope.

In another embodiment of the present invention, the recombinantKGF_(des1-23) polypeptide can be expressed as a fusion protein in any ofthe above expression systems. For example, for yeast expression, in theconstruction of an expression vector, a DNA sequence encoding theN-terminal portion of an endogenous yeast protein, or other stableprotein, can be fused to the 5' end of the coding sequence ofKGF_(des1-23). The DNA sequence at the junction of the two amino acidsequences optionally encodes a cleavable site, as discussed in EuropeanPatent Application, Publication No. 196 056.

An example of a DNA sequence that encodes the N-terminal portion of thefusion protein is the sequence encoding at least a portion of yeast orhuman superoxide dismutase ("SOD") for expression in yeast. Anotherexample is the sequence encoding at least a portion of the ubiquitinprotein, preferably, containing the sequence that encodes the cleavagesite or its processing enzyme, the ubiquitin-specific processingprotease. See, e.g., PCT Application Publ. No. WO 88/024066.

KGF toxin conjugates suitable for use herein can be produced by methodsknown in the art, for example, U.S. Pat. Nos. 4,771,128, 4,808,705, and4,894,443 and PCT Application, Publ. No. WO 92/04918.

The present invention includes variants and modifications of the abovethat do not substantially alter the nature and activity of the fragment,conjugate, therapeutic composition, vector, host, and methods of use andthat are apparent to a person of ordinary skill in the art.

EXAMPLES

The examples presented below are provided to demonstrate the presentinvention, and are not to be construed as limiting the invention in anyway.

Example 1

The Production of a KGF Coding Sequence Linked to a Signal Peptide

PCR techniques were applied to the construction of the KGF₁₆₃ codingsequence linked to a signal peptide, SEQ ID NO: 1. The signal peptidewas one that was associated with the native KGF₁₆₃ coding sequence, asdescribed in Finch et al., Science 245:752-755 (1989).

The following primers were used to clone the KGF₁₆₃ coding sequence andits signal peptide from a human kidney cDNA library: ##STR1##

As shown in the sequences above, in addition to the KGF₁₆₃ codingsequence and the signal peptide, additional nucleotides wereincorporated at the 5' and the 3' ends, respectively. The additionalnucleotides include a PstI site at the 5' end, and a NotI site at the 3'end of the respective primers, to facilitate cloning of the KGF₁₆₃ acoding sequence and its signal peptide into the desired insect celltransfer vector. (See Example 2 below.)

To generate the desired KGF₁₆₃ DNA molecule, 1 μl of each primer, at afinal concentration of 1 μM, was added to 2.5 μl or approximately 10 ng,of human kidney cDNA library, obtained from Clontech, Palo Alto, Calif.,U.S.A. The following reagents from the Perkin-Elmer PCR kit (Norwalk,Conn., U.S.A.) were also added to the primer/cDNA library: 16 μl of 1.25mM dNTP, containing equimolar amounts of each of dATP, dTPP, dCTP anddGTP, 10 μl of 10x buffer, 0.5 μl of Taq polymerase, at 5 units/ml; and69 μl of water. PCR was performed on a DNA Thermal Cycler, from PerkinElmer, Norwalk, Conn., U.S.A., as follows: 94° C. for 1 minute, 55° C.for 2 minutes, and 72° C. for 3 minutes. This temperature cycle wasrepeated 30 times. The resulting PCR product was treated with DNA PolI(Klenow) and then phenol/chloroform extracted, chloroform extracted, andethanol precipitated, respectively.

The PCR product was digested with PstI and NotI and gel purified beforeligation.

Example 2

The Production of a KGF Insect Cell Expression Vector

The purified PCR product, comprising the KGF₁₆₃ coding sequence and itssignal peptide, was inserted into a baculovirus transfer vector. Thespecific baculovirus used is Autographa californica nuclearpolyhedrosisvirus (AcNPV).

The purified PCR product from Example 1, digested with restrictionenzymes PstI and NotI, was ligated to pAcC13, which was also digestedwith PstI and NotI. pAcC13 is a baculovirus transfer vector and isdepicted in FIG. 2. This plasmid was derived from pAcC12, as describedin Munemitsu et al., Mol. Cell. Biol. 10:5977-5982 (1990) and aderivative of the pVL941 transfer vector, the construction of which isdescribed in Quilliam et al., Mol. Cell. Biol. 10:2901-2908 (1990);Luckow et al., Virol. 170:31-39 (1989); and Smith et al., Mol. Cell.Bio. 3(12): 2156-2165 (1983).

The transfer vector further contains a polylinker conveniently placedbetween the baculovirus polyhedrin promoter and terminator. The DNAsequence of the polylinker comprises unique restriction sites. Thus, bydigesting the transfer vector with the PstI and NotI sites within thepolylinker, the KGF₁₆₃ coding sequence and the signal peptide can beinserted between the polyhedrin promoter and terminator. In addition,the transfer vector contains sequences from the essential gene of AcNPVbaculovirus. The transfer vector was named pAcc/KGF.

Example 3

The Production of a Recombinant Baculovirus Capable of Expressing theKGF Coding Sequence

The transfer vector with the KGF₁₆₃ coding sequence and its signalpeptide insert was transfected together with a mutant baculovirus intoSpodoptera frugiperda, SF9 cells. The mutant baculovirus is a derivativeof AcNPV that lacks a functional essential gene. This mutant baculovirusmust recombine with the transfer vector to produce a viable baculovirus.To increase the number of recombinants, the mutant baculovirus waslinearized with BsuI. In this regard, several BsuI sites wereincorporated into the mutant baculovirus for this purpose. This mutantbaculovirus is similar to the baculovirus described in Kitts et al.,Biotechniques 14(5):810-817 (1993).

A. Preparation of Recombinant Baculovirus

First, 1×10⁶ SF9 cells were seeded per well in a 6-well plate containing2 ml of a complete TNMFH medium. The cells were incubated for at least30 minutes at room temperature to allow the cells to attach to theplate. The complete TNMFH medium contained GRACE'S medium obtained fromJRH Biosciences, Lenexa, Kans., U.S.A. and supplemented with 10% (v/v)fetal bovine serum (56° C. heat inactivated for 30 minutes), 3% (w/v)Yeastolate (Difco, Detroit, Mich., U.S.A.), and 1% (v/v) Fungi-BACT(Irvine Scientific, Santa Ana, Calif., U.S.A.).

For addition to each of the above wells, the following transfectionmixture was initially separately prepared by first, adding 0.5 ml ofGRACE'S medium containing no supplement into a sterile 1.5 ml eppendorftube; next, adding 0.5 μg of linearized mutant baculovirus DNA and,approximately, 2-3 μg of the transfer vector containing the KGF₁₆₃coding sequence and the signal peptide insert. This is approximately a4:1 ratio of transfer vector to mutant baculovirus. Finally, thecationic liposome solution, BRL catalog #8282SA (from BRL, Gaithersburg,Md., U.S.A.) was mixed thoroughly, and 10 μl of this liposome solutionwere added to the baculovirus mixture. This transfection mixture wasincubated at room temperature for 15 minutes.

Before the transfection mixture was added to the cells in the wells, theTNMFH medium was removed therefrom, and the cells were washed with 1-2ml of GRACE'S medium without supplements. When the transfection mixturewas finally prepared, as described above, all the media were removedfrom the SF9 cells, and the transfection mixture was added dropwise tothe cells. The 6-well plate was then covered with parafilm to reduceevaporation of the transfection mixture. The plate was rocked slowly atroom temperature for approximately four hours on a Bellco®, catalog no.#774020020, Vineland, N.J., U.S.A., side/side rocking platform atsetting 2.5.

After the incubation of the cells with the transfection mixture, 0.5 mlof complete TNMFH medium was added to the cells and the mixture wasincubated at 27° C. in a humidified chamber (92%) for 48 hours.Thereafter, the medium containing recombinant baculovirus was removedfrom the cells and stored at 4° C. until the plaque assay forrecombinant virus isolation was performed. This medium constituted theprimary source of the recombinant virus.

After the medium was removed from the cells, 2 ml of complete TNMFH wereadded to the cells, and the cells were further incubated in a humidifiedchamber (92%) at 27° C. for another 48-72 hours. This final step wasperformed to provide a back-up source of recombinant virus and toprovide a visual record of the viral infection.

B. Plaque Purification of the Recombinant Baculovirus

The recombinant KGF baculovirus prepared as above were plaque-purifiedaccording to the following steps:

First, 4 ml of SF9 cells at 5×10⁵ cells/ml in GRACE'S medium were platedon 60 mm LUX dishes, catalog #5220. The cells were incubated at roomtemperature for 20-30 minutes to allow the cells to adhere to the plate.In the meantime, the medium containing the primary source of recombinantvirus was diluted into 2 ml of TNMFH medium at 1:10, 1:50, 1:100, and1:200 dilutions. After the cells were allowed to adhere, the medium wasaspirated from the adherent cells, and the various dilutions of therecombinant virus were added quickly so as not to allow the cells todry. The cells were then incubated for 1 hour at 27° C. in a humidifiedchamber (92%).

Ten minutes before the incubation was complete, an agarose solution wasprepared. A 2x concentration of GRACE'S medium supplemented as beforewas heated to 37° C. Only the amount used for the assay was heated;otherwise, proteins may precipitate upon repeated heating. When the 2xmedium was warm, a 3% (w/v) Sea Plaque agarose mixture in water wasmelted in a microwave and immediately mixed 1:1 with the 2x medium. Theagarose solution was then allowed to cool at room temperature forseveral minutes.

The viral medium was aspirated from the cells in the dishes by tiltingthe dishes slightly on the hood rim. The dishes were then drained for afew seconds and aspirated again to remove as much liquid as possible.This second aspiration step was included to reduce the likelihood ofvirus spreading and causing indistinct plaques. Six dishes or fewer werehandled at a time to avoid drying the cells. The aspirated dishes werenever left exposed with the lids off for more than a few seconds.

Next, 4 ml of the agarose solution were added to each dish and leftundisturbed for 15 minutes. The agarose overlay was dried by lifting thelids to the side of the plate to permit the agarose to dry forapproximately 25 minutes. Then, the dishes were covered with the lids,and the cells were incubated in a humidified chamber (92%) for 4 days at27° C.

To facilitate visualization of the plaques, the dishes were stained with2 ml per dish of 25% (w/v) Sea Plaque agarose in complete TNMFH mediumwith 0.01% (v/v) neutral red, from Sigma, St. Louis, Mo., U.S.A. Theagarose overlay was dried at room temperature for about one hour withthe lids on, before the dishes were returned to a humidified chamber(92%) at 27° C. for 3-4 hours. The neutral red dye was incorporated bythe viable but not the dead cells.

When the plaques were well-resolved, 7 individual plugs were picked witha sterile Pasteur piper and each was transferred to 1 ml of completeTNMFH. The plugs were incubated for 2 days at room temperature. Theplugs were then vortexed, and the plaque assay was repeated with 50 μlof this solution.

C. Expansion of the Plaque Purified Baculovirus

To expand the viral titers, 3-4 plugs of each baculovirus clone from thesecond round of plaque purification were placed directly onto culturesof SF9 cells. The cells were plated at 2.5×10⁵ /well in 6-well plateswith 2.5 ml of complete TNMFH medium. The cells and virus were thenincubated for approximately 4 hours at 27° C. in a humidified chamber(92% ).

For the second round of expansion, all the virus from the 6-well plateswere transferred to 10 cm dishes. The 10 cm dishes were plated with7.5×10⁶ cells in 7.5 ml of complete TNMFH media. The cells and viruswere incubated for 48-72 hours at 27° C. in a humidified chamber (92%).

After this infection, the cells were thoroughly screened for any wildtype virus contamination that appeared as infected cells containingocclusion bodies. No occlusion bodies were observed. The resultingrecombinant baculovirus was named KGF-5 and was deposited with theAmerican Type Culture Collection ("ATCC") at 12301 Parklawn Drive,Rockville, Md., U.S.A. 20852-1776 on 17 Jun. 1993, Accession No. VR2411.

Supernatant from the second baculovirus expansion round was tested forKGF bioactivity. Also, the crude supernatant was run on a sodium dodecylsulfate polyacrylamide gel electrophoresis ("SDS-PAGE") gel, and stainedwith Coomassie blue to confirm the expression of KGF.

Example 4

Baculovirus Expression of a KGF Coding Sequence

SF9 insect cells were infected with the recombinant baculovirus, asdescribed in Example 3, to provide a large of amount of recombinant KGFfragments for purification and analysis.

More specifically, SF9 cells were diluted to a cell density of 1×10⁶cells/ml in Excell-400 (JRH Biosciences, Lenexa, Kans. U.S.A.). Thecells were seeded into either a 1 L or 2 L shake flask with 300 ml or500 ml of medium, respectively. Virus from the second expansion(described in Example 3, section C) was used to inoculate the cells at10% (v/v) dilution. The cells and virus were shaken at approximately 131rpm with the caps of the shake flasks loosened to provide an adequateair supply. The cells were incubated at 27° C. for 48 hrs.

Conditioned medium from the incubated cell culture above was collectedby centrifuging the cell culture at 15,000×g for 10 minutes at 4° C. toremove the cells. Next, the conditioned medium was filtered with an 0.8μm cellulose nitrate filter (Millipore). Approximately 5 liters of thisconditioned medium was concentrated to 200 ml using a filtron cassettesystem (Omega membrane), having a 3 kD molecular weight cut-off. The pHof the retentate was adjusted to 7.2 with 1N NaOH and the small amountof precipitate was removed by centrifugation at 10,000×g for 20 minutesat 4° C.). The concentrated conditioned medium was then immediatelyapplied to a Heparin Sepharose® ("HS") resin column.

Example 5

Purification of a KGF_(des1-23) Fragment

Recombinant KGF_(des1-23) from the concentrated conditioned mediumdescribed in Example 4 was purified by HS affinity chromatography,followed by Mono S cation exchange chromatography. The recombinantKGF_(des1-23) bound to the HS was eluted using step-wise salt gradient.Next, the recombinant KGF_(des1-23) eluted from the HS column was boundto the Mono S cation column and was eluted using a linear salt gradient.

Further details of the procedure utilized are described below.

A. Heparin Sepharose® Affinity Chromatography

First, the concentrated conditioned medium from Example 4 was allowed torun for approximately 2 hours at 4° C. through a 30 ml bed of HS resin.The column was equilibrated in a buffer containing 150 mM NaCl, and 10mM Tris-HCl at pH 7.3. Once the concentrated conditioned medium wasloaded, the column was washed extensively with the equilibration bufferuntil the absorbance at 280 nm returned to baseline. Then, protein waseluted from the HS column with an increasing step-wise NaCl gradient.The NaCl concentrations were 0.45M, 1M, and 2M NaCl, in 10 mM Tris-HClat pH 7.3. The flow rate of the column during elution was approximately90 ml/hr and 3-ml size fractions were collected.

The fractions were tested for KGF bioactivity utilizing Balb/Mk cells.The assay is described in section C below. The fractions with thehighest bioactivity were eluted with 1M NaCl and were pooled. Before thepooled fractions were loaded onto the next column, the fractions werediluted five-fold with 10 mM Tris-HCl at pH 7.2 to a final saltconcentration of 0.2M NaCl.

B. Mono S Cation Exchange Chromatography

The pooled fractions eluted from the HS column were loaded with a Superloop onto a Mono S column linked to an FPLC system (Pharmacia,Piscataway, N.J.). The Mono S cation exchange column was equilibratedwith 10 mM Tris-HCl at pH 7.2. When the pooled fractions were loaded,the column was washed extensively at a flow rate of 1 ml/min. with theequilibration buffer until the absorbance returned to baseline. Then,protein was eluted from the column with a linear NaCl gradient, 0.2M to1M NaCl in 10 mM Tris-HCl at pH 7.3 at a flow rate of 1 ml/min., and 1ml fractions were collected.

Two major protein peaks of activity were found that eluted at about0.55M NaCl and 0.60M NaCl . Fractions across the protein peaks wereassayed for bioactivity and subjected to SDS-PAGE analysis. The resultsof the gel analysis (not illustrated) showed that protein from 0.55M and0.60M NaCl fractions exhibited an apparent molecular weight of 27 kD and18 kD, respectively.

When the bioactivity of the fractions was determined, the 18 kD proteinwas found to exhibit at least a 2-, but more particularly a 7-10 foldmore activity than the 27 kD protein, or than the positive control, theacidic fibroblast growth factor ("aFGF"), as shown in FIG. 5. Thebioactivity of the fractions were tested using Balb/Mk cells. (Seesection C, below.)

To determine the amino acid sequence of the eluted protein, two 100pmole samples of the 27 kD and 18 kD proteins were subjected to Edmandegradation after centrifugal adsorption to polyvinylidene difluoride(PVDF, Applied Biosystems Prospin). The samples were loaded onto anApplied Biosystems 470A or 473A protein sequencer. Twenty rounds ofEdman degradation were carried out using standard software and chemicalssupplied by Applied Biosystems, and identifications of PTH-amino acidswere made with an automated on-line HPLC system (FFH analyzer 120A,Applied Biosystems).

Using standard methodology well known in the art, an unambiguous aminoacid sequence was established for positions 1 to 20 of the N-terminal ofthe 18 kD protein, which is KGF_(des1-23). The sequence was as follows:

S Y D Y M E G G D I R V R R L F X R T Q (SEQ ID NO: 7). The N-terminalsequence of the 27 kD protein is identical to the N-terminal sequence ofKGF₁₆₃.

C. KGF Bioactivity Assay Utilizing Balb/Mk Cells

KGF bioactivity was assessed by the ability of the fractions to promotegrowth of BALB/C-Mk cells.

Stock cultures of Balb/Mk cells were grown and maintained in low calciumDulbecco's modified Eagle medium supplemented with 10% fetal bovineserum, 0.25 μg/ml fungizone, and 10 ng/ml aFGF. The cells were incubatedat 37° C. in a 10% CO₂ atmosphere with 99% humidity. For the bioactivityassay, the cells were seeded in 12-well plates at a density of 5×10³cells per well in 1 ml of medium as described above for the stockcultures, and as described in Gospodarowicz et al., J. Cell. Physiol.142: 325-333 (1990).

Ten microliter aliquots of the desired column fractions were dilutedinto 1 ml of 0.2% (w/v) gelatin in phosphate buffered saline ("PBS").Ten microliters of this dilution were added to Balb/Mk cells seeded in12-well cluster plates containing 22 mm webs, at 5×10³ cells per well,and a 10 μl aliquot of either the diluted column fractions or mediumcontaining 10 ng aFGF were added to the cells every other day.

After five days in culture, the cells were trypsinized and the finalcell density was determined using a Coulter® counter. The cells werereleased from the plates by replacing the culture medium with a solutioncontaining 0.9% NaCl, 0.01M sodium phosphate (pH 7.4), 0.05% trypsin,and 0.02% EDTA (STV). The cells were incubated in this solution for 5-10minutes at 37° C., and then the stock culture medium was added to thecells. The cells were then counted using a Coulter® counter (CoulterElectronics, Hialeah, Fla., U.S.A.). Results shown in FIG. 4demonstrates the effect of different dilutions of rKGF_(des1-23) andrKGF₁₆₃ on Balb/Mk cells as compared to aFGF. The figure legend for FIG.4 is as follows: ◯=KGF_(des1-23), =KGF₁₆₃, and □=aFGF.

The final cell density was graphed as a function of proteinconcentration. The protein concentration is graphed on a log scale. Theprotein concentration was determined by Bradford assay according toinstructions accompanying the protein assay kit from BIORAD (Richmond,Calif., U.S.A.).

The ED₅₀ was calculated by (a), dividing in half the difference betweenthe lowest and highest cell density value of the curve; and (b),determining from the graph what protein concentration corresponds tothat cell density number obtained in (a). According to FIG. 4, the ED₅₀for aFGF was found to be approximately 500 pg/ml, that for rKGF₁₆₃ wasabout 250 pg/ml, and that for KGF_(des1-23) was about 24 pg/ml. Theresults of this assay demonstrate that rKGF_(des1-23) was about 10-foldmore active than rKGF₁₆₃.

The exact ED₅₀ for the proteins may vary from assay to assay, but theratio of the activity between the long and short form of KGF is at least2 fold, preferably, at least a 7-fold, more preferably, about 7-10 fold,even more preferably, at least 10 fold. Even a 50-fold difference inactivity had been observed.

For example, the bioactivity assay was repeated with different or withthe same preparations of rKGF₁₆₃ and rKGF_(des1-23). The results of oneof the assays showed that the ED₅₀ of rKGF₁₆₃ and rKGF_(des1-23) were600 pg/ml, and 80 pg/ml, respectively, about a 7-fold increase inactivity. Another assay demonstrated a 50-fold difference between thelong and short form of KGF. In this later assay, KGF_(des1-23) exhibitedan ED₅₀ of 30 pg/ml, and KGF₁₆₃ exhibited an ED₅₀ of 1500 pg/ml. In viewof the fact that the last bioassay was done two (2) months after KGFisolation, the data suggest that upon storage, KGF₁₆₃ lost itsbioactivity faster than KGF_(des1-23).

D. Absence of KGF Bioactivity on ABAE or ACE Cells

KGF can be characterized by its lack of activity on vascular endothelialcells derived from large vessels ("ABAE") or capillary cells ("ACE") ascompared with basic FGF ("bFGF"). Stock cultures of ABAE and ACE cellswere grown and maintained in low calcium Dulbecco's modified Eaglemedium supplemented with 10% bovine serum, 0.25 μg/ml fungizone, and 2ng/ml bFGF. The cells were incubated at 37° C. with a 10% CO₂concentration and 99% humidity.

In the mitogenic assay, either 10⁴ ABAE or 5×10³ ACE cells were platedper well in 12-well plates in stock culture medium, as described isGospodarowicz et al., Proc. Natl. Acad. USA 73:4120-4124 (1976);Gospodarowicz et al., J. Cell. Physiol. 127:121-136 (1976); andGospodarowicz et al., Proc. Natl. Acad. USA 86:7311-7315 (1989). Tenmicroliter aliquots of the fractions to be tested were diluted into 1 mlof 0.2% (w/v) gelatin in phosphate buffered saline ("PBS"). Tenmicroliters of this dilution was added to cells seeded in 12-wellcluster plates. A 10 μl aliquot of either the diluted purified rKGFmedium containing fractions or 2 ng bFGF was added every other day.

After five days in culture, the cells were trypsinized and the finalcell density was determined using a Coulter® counter. The cells werereleased from the plates by replacing the medium with a solutioncontaining 0.9% NaCl, 0.01M sodium phosphate (pH 7.4), 0.05% trypsin,and 0.02% EDTA (STV). The cells were incubated in this solution for 5-10minutes at 37° C., and then the stock culture medium was added to thecells. The cells then were counted using a Coulter® counter (CoulterElectronics, Hialeah, Fla., U.S.A.).

Results shown in FIG. 5 demonstrates the lack of activity of bothrKGF₁₆₃ and rKGF_(des1-23) on ABAE cells and ACE cells, in contrast tobFGF. Similar observations were made using vascular smooth muscle cells,corneal endothelial cells, ovarian grandulosa cells, and BkH-21fibroblasts. Although bFGF did stimulate the proliferation of thesevarious cell types, neither rKGF₁₆₃ or rKGF_(des1-23) had anybioactivity.

Example 6

Construction of a KGF_(des1-23) Yeast Vector for Secretion by YeastCells

This example describes the construction of a yeast expression vector forsecretion of KGF_(des1-23) by transformed yeast cells in accordance withthe present invention.

Specifically, the components of the expression vector construct:KGF_(des1-23) coding sequence operably linked, at its 5' end, to aglyceraldehyde-3-phosphate dehydrogenase ("GAPDH") promoter and thecoding sequence of the first 35 amino acid residues of the Saccharomycescerevisae prepro α-factor leader sequence and, at its 3' end, to theSaccharomyces cerevisae α-factor terminator.

The fragments for this construct:

(1) a BamHI/Bpu1102I fragment containing the GAPDH promoter and thetruncated prepro α-factor leader containing a functional signal peptide;

(2) a Bpu1102I/SalI fragment encoding KGF_(des1-23) ; and

(3) a BamHI/SalI vector fragment of the yeast expression vector,pBS24.1, containing the α-factor terminator, leucine, and uracil yeastselectable markers, and 2μ sequences as an origin of replication.

A. Construction of the BamHI/Bpu1102I Promoter/Leader Fragment

The source of the BamHI/Bpu1102I fragment: by PCR using pGAI7 as atemplate. The plasmid pGAI7 is described in European Patent Application,Publication No. 324 274. This plasmid contains the same BamHI expressioncassette as pYGAI7, which is available at the ATCC as Accession No.67597. Plasmid pGAI7 contains approximately 400 bp GAPDH promoterfragment, as described in Travis al., J. Biol. Chem. 4384-4389 (1985),and the prepro α-factor leader sequence that encodes the N-terminalamino acid residues 1-35.

The primer for the PCR of BamHI/Bpu1102I promoter/leader fragment:##STR2##

Reagents for generating the BamHI/Bpu1102I fragments by PCR: 1 μl ofeach primer (100 pmoles/μl); 1 μl of the template, pGAI7 at ˜500 μg/ml,and the following reagents from Perkin Elmer PCR kit (Norwalk, Conn.): 8μl of dNTP (1.25 mM each of dATP, dTPP, dCTP, and dGTP), 10 μl of 10xbuffer, 0.5 μl of Taq polymerase, and 63.5 μl of water. The temperaturecycle for PCR: 94° C. for 1 minute, 37° C. for 2 minutes, and 72° C. for3 minutes. Number of repetitions of this temperature cycle: 30.Purification of the PCR product of ˜414 bp by gel purification, and bythe use of a Geneclean kit by bio101, La Jolla, Calif., U.S.A.

B. Construction of the Bpu1102I/SalI KGF_(des1-23) Coding SequenceFragment

The origin of the Bpu1102I/SalI KGF_(des1-23) coding sequence fragment:by PCR using the pAcc/KGF baculovirus transfer vector described inExample 1 and 2.

The primers for PCR of the Bpu1102I/SalI KGF_(des1-23) coding sequence:##STR3##

Reagents for generating the Bpu1102I/SalI KGF_(des1-23) coding sequenceby PCR: 1 μl of each primer (100 pmoles/μl), 1 μl of the template,pAcc/KGF, at 500 μg/ml, and the following reagents from Perkin Elmer PCRkit (Norwalk, Conn., U.S.A.): 8 μl of dNTP (1.25 mM each of dATP, dTYP,dCTP, dGTP), 10 μl of 10x buffer, 0.5 μl of Taq polymerase, and 63.5 μlof water. The temperature cycle for PCR: 94° C. for 1 minute, 37° C. for2 minutes, and 72° C. for 3 minutes. Number of repetitions of thistemperature cycle: 30. Purification of the PCR product of ˜414 bp by gelpurification, and by use of a Geneclean kit by bio101, La Jolla, Calif.,U.S.A.

C. Construction of the BamHI/SalI Vector Fragment

The source of the BamHI/SalI vector fragment: by digesting the yeastexpression vector pBS24.1 with BamHI and SalI.

pBS24.1 is a yeast expression vector that is derived from pAB24. pBS24.1contains the α-factor terminator; the 2μ sequences, as a yeast origin ofreplication; selectable markers for uracil and leucine in yeast; and anampicillin resistance gene, effective in E. coli. This yeast expressionvector also contains a human FGF coding sequence which can be excised bydigestion with BamHI and SalI enzymes and isolating the larger vectorfragment.

Plasmid pAB24 is a yeast shuttle vector that contains the complete 2μsequence, as described in Broach, Molecular Biology of the YeastSaccharomyces, Cold Spring Harbor Press 1:445 (1981) and pBR322sequences. It also contains the yeast URA3 gene derived from plasmidYEp24, as described in Botstein, et al., Gene 8:17 (1979) and the yeastLEU^(2d) gene derived from plasmid pC1/1, as described in EuropeanPatent Application, Publication No. 116 201. Plasmid pAB24 wasconstructed by digesting YEp24 with EcoRI and religating the vector toremove the partial 2μ sequences. The resulting plasmid, YEp24ΔR1, waslinearized by digestion with ClaI and ligated with the complete 2μplasmid which had been linearized with ClaI. The resulting plasmid,pCBou, was then digested with XbaI and the 8605 bp vector fragment wasgel isolated. This isolated XbaI fragment was ligated with a 4460 bpXbaI fragment containing the Leu^(2d) gene isolated from pC1/1; theorientation of the LEU^(2d) gene is in the same direction as the URA3gene. Insertion of the expression cassette was in the unique BamHI siteof the pBR322 sequences, this interrupting the gene for bacterialresistance to tetracycline.

Plasmid pBS24 is a derivative of pAB24 as described. Plasmid pAB24 wasdigested with BamHI and SalI, which cut within the tetracycline gene ofthe pBR322 sequences, and gel purified. The vector was then ligated witha synthetic adapter of the following sequence which created new uniqueBglII and BamHI sites: ##STR4##

The resulting vector, pAB24ΔBL was then digested with BamHI and BglIIand gel purified. The linearized vector was ligated with the BamHIcassette excised and purified from pSOD/env-5b to give pBS24. Thecassette contains the hybrid ADH-2/GAPDH promoter and α-factorterminator with an NcoI-SalI insert of the SOD/env-5b fusion gene. Thecassette is oriented in pBS24 such that the direction of transcriptionfrom the ADH-2/GAPDH promoter is in the opposite direction to that ofthe inactivated tetracycline gene of the pER322 sequences.

pBS24.1 contains a BamHI/SalI fragment encoding the human FGF proteininstead of the SOD/env-5b in pBS24, as described in U.S. Pat. No.5,156,949. The relevant vector fragment of pBS24.1 is present inpBS24.1bBMP. pBS24.1bBMP is the same as pBS24.1 except is contains aBamHI/SalI bovine BMP expression cassette instead of the human FGFprotein. The vector pBS24.1bBMP was deposited with the ATCC, Rockville,Md., U.S.A., on 1 Jun. 1989 under Accession no. 20949.

Example 7

Expression of rKGF_(des1-23) by Yeast Cells

The procedure for expression of rKGF_(des1-23) in accordance with thepresent invention:

The secretory expression vector: as described in Example 6.

Host for expression: Saccharomyces cerevisae.

Method of introducing the expression vector into host cells: byelectroporation, as described in "Guide to Yeast Genetics & MolecularBiology," in Methods in Enzymology, Vol. 194 (Academic Press 1991).

Selection of transformants: on ura with 2% glucose medium.

Seed culture of transformants: overnight incubation in 1 ml of leu with2% glucose medium at 30° C. in a shaking apparatus.

For production of rKGF_(des1-23) expression: 20 ml culture seeded withthe overnight culture in ura with 2% glucose medium, for approximately72 hours.

Example 8

Construction of a KGF_(des1-23) Yeast Vector for Intracellular YeastExpression

This example describes a procedure for construction of a yeastexpression vector, for intracellular expression of the KGF_(des1-23) bytransformed yeast cells, in accordance with the present invention:

Specifically, the components of the expression vector: KGF_(des1-23)coding sequence, a hybrid yeast promoter and an α-factor terminator.

The fragments for construction of this vector:

(1) BamHI/NcoI fragment containing the ADH2/GAPDH promoter;

(2) BspHI/SalI fragment encoding KGF_(des1-23) ; and

(3) BamHI/SalI vector fragment of the yeast expression vector, pBS24.1,containing the Saccharomyces cerevisae α-factor terminator, leucine anduracil yeast selectable markers, and 2μ sequences as an origin ofreplication.

A. Construction of a KGF_(des1-23) Coding Sequence

Construction of the BspHI/SalI KGF_(des1-23) coding sequence fragment:by PCR using the pAcc/KGF baculovirus transfer vector, as described inExample 1 and 2.

The primers for PCR: ##STR5##

Reagents for construction of the BspHI SalI KGF_(des1-23) codingsequence by PCR: 1 μl of each primer (100 pmoles/μl), 1 μl of thetemplate, pGAI7, at ˜500 μg/ml, and the following reagents from PerkinElmer PCR kit (Norwalk, Conn.): 8 μl of dNTP (1.25 mM each of dATP,dTPP, dCTP, and dGTP), 10 μl of 10x buffer, 0.5 μl of Taq polymerase,and 63.5 μl of water. The temperature cycle for PCR: 94° C. for 1minute, 37° C. for 2 minutes, and 72° C. for 3 minutes. Number ofrepititions of the temperature cycle: 30. Purification of the PCRproduct of ˜414 bp by gel purification, and by use of a Geneclean kit bybio101, La Jolla, Calif., U.S.A.

B. Construction of a BamHI/NcoI Promoter Fragment

The source of the BamHI/NcoI promoter fragment: by digestion of pSI3containing a superoxidase dismutase (SOD)/insulin fusion yeastexpression cassette comprising a BamHI/NcoI, ADH2/GAPDH hybrid promoter,to release a fragment of about 1366 bp containing the hybrid promotersequences.

pSI3 is a derivative of pYASI1, which was deposited with the ATCC,Rockville, Md., U.S.A, on 27 Feb. 1985, and assigned Accession no.20745. The construction of pYASI1 is described in U.S. Pat. No.4,751,180. pYASI1 contains the same BamHI/NcoI hybrid promoter fragmentas pSI3.

C. Construction of the BamHI/SalI pBS24.1 Vector Fragment

Source of BamHI/SalI pBS24.1 vector fragment: by digestion of pBS24.1with BamHI and SalI and isolation of the large vector fragment, asdescribed in Example 6, section C.

Example 9

Intracellular Expression of rKGF_(des1-23) by Yeast Cells

Procedure for intracellular expression of rKGF_(des1-23) by yeast cellsin accordance with the present invention:

Expression vector for intracellular expression in yeast cells: asdescribed in Example 8.

Host cells for transformation: Saccharomyces cerevisae.

Method of introducing expression vector into host cells: byelectroporation, was described in "Guide to Yeast Genetics & MolecularBiology," Methods in Enzymology, Vol. 194 (Academic Press, 1991).

Selection of transformants: on ura with 2% glucose medium.

Seed culture of transformants: overnight incubation in 1 ml of leu with2% glucose medium at 30° C. in a shaking apparatus. Culture forproduction of rKGF_(des1-23) : a 20 ml culture seeded with the overnightculture in ura with 2% glucose medium for approximately 72 hours.

Deposit Information:

The following materials were deposited with the American Type CultureCollection:

    ______________________________________                                        Virus Name         Deposit Date                                                                             Accession No.                                   ______________________________________                                        Autographa californica                                                                           17 Jun 1993                                                nuclearpolyhedrosis virus KGF-5                                               VR2411                                                                        Escherichia coli, pYGAI7                                                                         29 Dec 1997                                                                              67597                                           Saccharomyces cerevisae                                                                          1 Jun 1989 20949                                           pBS24.1bBMP                                                                   Saccharomyces cerevisae, 2150-2-3                                                                27 Feb 1985                                                                              20745                                           pYASI1                                                                        ______________________________________                                    

The above materials have been deposited with the American Type CultureCollection, Rockville, Md., under the accession numbers indicated. Thisdeposit will be maintained under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for purposesof Patent Procedure. The deposits will be maintained for a period of 30years following issuance of this patent, or for the enforceable life ofthe patent, whichever is greater. Upon issuance of the patent, thedeposits will be available to the public from the ATCC withoutrestriction.

These deposits are provided merely as convenience to those of skill inthe art, and are not an admission that a deposit is required under 35U.S.C. §112. The sequence of the polynucleotides contained within thedeposited materials, as well as the amino acid sequence of thepolypeptides encoded thereby, are incorporated herein by reference andare controlling in the event of any conflict with the writtendescription of sequences herein. A license may be required to make, use,or sell the deposited materials, and no such license is granted hereby.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 19                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 194 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetHisLysTrpIleLeuThrTrpIleLeuProThrLeuLeuTyrArg                              151015                                                                        SerCysPheHisIleIleCysLeuValGlyThrIleSerLeuAlaCys                              202530                                                                        AsnAspMetThrProGluGlnMetAlaThrAsnValAsnCysSerSer                              354045                                                                        ProGluArgHisThrArgSerTyrAspTyrMetGluGlyGlyAspIle                              505560                                                                        ArgValArgArgLeuPheCysArgThrGlnTrpTyrLeuArgIleAsp                              65707580                                                                      LysArgGlyLysValLysGlyThrGlnGluMetLysAsnAsnTyrAsn                              859095                                                                        IleMetGluIleArgThrValAlaValGlyIleValAlaIleLysGly                              100105110                                                                     ValGluSerGluPheTyrLeuAlaMetAsnLysGluGlyLysLeuTyr                              115120125                                                                     AlaLysLysGluCysAsnGluAspCysAsnPheLysGluLeuIleLeu                              130135140                                                                     GluAsnHisTyrAsnThrTyrAlaSerAlaLysTrpThrHisAsnGly                              145150155160                                                                  GlyGluMetPheValAlaLeuAsnGlnLysGlyIleProValArgGly                              165170175                                                                     LysLysThrLysLysGluGlnLysThrAlaHisPheLeuProMetAla                              180185190                                                                     IleThr                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CysAsnAspMetThrProGluGlnMetAlaThrAsnValAsnCysSer                              151015                                                                        SerProGluArgHisThrArg                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetHisLysTrpIleLeu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AGATCTCTGCAGCTATAATGCACAAATGGATACTG35                                         (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ThrIleAlaMetProLeuPhe                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AGATCTGCGGCCGCTTAAGTTATTGCCATAGGAAGAAA38                                      (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       SerTyrAspTyrMetGluGlyGlyAspIleArgValArgArgLeuPhe                              151015                                                                        XaaArgThrGln                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GGTGGTGGATCCCCAGCTTAGTTCATAGGTCC32                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       HisGlnAsnValPheArgLysAlaProIleGlnAla                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GTGTTGGTTAACGAATCGCTTAGCCGGAATTTGTGC36                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ProAlaLysArgSerTyrAspTyrMetGluGlyGly                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CCGCCGGCTAAGCGAAGTTATGATTACATGGAAGGAGGG39                                     (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ThrIleAlaMetProLeuPheHis                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GGTGGTGTCGACTTAAGTTATTGCCATAGGAAGAAAGTG39                                     (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GATCAGATCTAAATTTCCCGGATCC25                                                   (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      TCTAGATTTAAAGGGCCTAGGAGCT25                                                   (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      MetSerTyrAspTyrMetGluGlyGly                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GTTGTTTCATGAGTTATGATTACATGGAAGGAGGG35                                         (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 99 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: -                                                               (B) LOCATION: 13..14                                                          (D) OTHER INFORMATION: /note= "The figure did not contain                     the intervening polyhedrin sequences."                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      TATAAATATTCCGGGCGCGGATCGGTACCAGATCTGCAGAATTCTAGAGGATCCTGATCA60                GCTAGCAGAGCTCGCGGCCGCCCGGGCCGTACCGACTCT99                                     __________________________________________________________________________

What is claimed:
 1. A keratinocyte growth factor fragment that exhibitsat least a 2-fold increase in mitogenic activity as compared to amature, recombinant, full-length keratinocyte growth factor, wherein thefragment lacks the first 23 N-terminal amino acid residues of themature, full-length keratinocyte growth factor but retains the remainderof the molecule.
 2. The keratinocyte growth factor fragment as claimedin claim 1, wherein the fragment exhibits a 7-fold increase in mitogenicactivity as compared to the mature, recombinant, full-lengthkeratinocyte growth factor.
 3. The keratinocyte growth factor fragmentas claimed in claim 1, wherein the fragment exhibits a 10-fold increasein mitogenic activity as compared to the mature, recombinant,full-length keratinocyte growth factor.
 4. The keratinocyte growthfactor fragment as claimed in claim 1, wherein the fragment exhibitsdecreased cytotoxicity as compared to the mature, recombinant,full-length keratinocyte growth factor.
 5. A composition comprising:(a)a keratinocyte growth factor fragment that exhibits at least a 2-foldincrease in mitogenic activity as compared to the mature, recombinant,full-length keratinocyte growth factor, wherein the fragment lacks thefirst 23 N-terminal amino acid residues of the mature, full-lengthkeratinocyte growth factor but retains the remainder of the molecule,and (b) a carrier.
 6. The keratinocyte growth factor fragment as claimedin claim 1 having the amino acid sequence depicted at amino acidresidues 24 to 163, inclusive, of FIG. 1 (amino acid residues 55-194,inclusive, of SEQ ID No: 1).
 7. An analog of the keratinocyte growthfactor fragment as claimed in claim 6, wherein the analog differs fromits keratinocyte growth factor fragment of claim 6 in having at leastone conservative substitution selected from the group of substitutionsconsisting of a substitution of a leucine with an isoleucine or anisoleucine with a leucine, a substitution of a leucine with a valine ora valine with a leucine, a substitution of an aspartic acid with aglutamic acid or a glutamic acid with an aspartic acid and asubstitution of a threonine with a serine or a serene with a threonineand further wherein the analog exhibits the biological activity of thekeratinocyte growth factor fragment.
 8. An analog of the keratinocytegrowth factor fragment as claimed in claim 6, wherein the analog differsfrom its keratinocyte growth factor fragment of claim 6 in having atleast one cysteine residue substituted by another amino acid and furtherwherein the analog exhibits the biological activity of the keratinocytegrowth factor fragment.
 9. The analog as claimed in claim 8, wherein thecysteine residue is replaced with serine or threonine.
 10. A compositioncomprising:(a) the keratinocyte growth factor fragment as claimed inclaim 6, and (b) a carrier.
 11. A composition comprising:(a) the analogas claimed in claim 7, and (b) a carrier.
 12. A compositioncomprising:(a) the analog as claimed in claim 5, and (b) a carrier. 13.A composition comprising:(a) the analog as claimed in claim 9, and (b) acarrier.